Avidin or streptavidin (hereinafter referred to as (strept)avidin) has highly specific affinity with biotin or any derivatives thereof. When the both are mixed with each other under a physiological condition, they are easily bound to form a complex (the complex between (strept)avidin and biotin or between (strept)avidin and the biotin derivative are generally called as “ABC complex”). This property is utilized for isolation of a target substance or a target material such as cells (Patent Literature 1).
A carrier for capturing a target substance may be prepared and the target substance may be captured by use of this carrier, for example, a biotin-labeled probe molecule (such as an antibody having binding affinity for the target substance), to which biotin is beforehand bound, are mixed with an insoluble carrier to which (strept)avidin is immobilized under a physiological condition, to form an ABC complex. Thereafter, if the ABC complex or the binding between the probe molecule and the target substance can be cleaved, the target substance can be isolated (Patent Literature 1).
However, the affinity of (strept)avidin for biotin is so strong that it is very difficult to cleave the ABC complex under physiological condition. In order to dissociate (strept)avidin from a biotin derivative, a severe condition, for examples, a condition wherein the combination is treated with a guanidine hydrochloride salt in pH of about 1.5, and a condition wherein the combination is boiled in a buffer solution for SDS polyacrylamide gel electrophoresis to which a reducing agent is added, is required. Accordingly, when a target to be isolated is a protein, cells, or other target substance that is easily damaged by a severe pH, heat or salt concentration, or physical shear force, it is generally difficult to isolate the target substance wherein the physiological activity thereof is maintained.
Therefore, methods for easily isolating a target substance, for example, a method using a biotin derivative which binds to (strept)avidin with lower affinity than biotin (Patent Literature 2); and a method using an avidin variant lower in affinity with biotin than avidin (Patent Literature 3), are proposed. For example, a method in which, instead of biotin, desthiobiotin or some other biotin derivative which binds to (strept)avidin with lower affinity than biotin (Patent Literature 2) is used to prepare a capturing carrier as described above, a target substance is captured thereby, and then a large amount of biotin is added thereto for cleaving the ABC complex to isolate the target substance. In this case, the biotin is thought to function as a competitive inhibitor for bonding between desthiobiotin and (strept)avidin. However, according to this method in which biotin is used as competitive inhibitor, it would be difficult to efficiently cleave the ABC complex. Thus, the target substance would be difficult to be efficiently isolated.